Method and composition for inhibiting arteriosclerosis

ABSTRACT

The present invention has for its object to provide a method and a composition for achieving a more efficient recovery of the antioxidant activity of plasma LDL, or even augmentation thereof, which has been depressed by the use of an antihyperlipidemic or cholesterol-lowering drug to thereby insure a potentiated antiarteriosclerotic efficacy. A method for inhibiting arteriosclerosis, which comprises using a combination of a cholesterol-lowering agent and a reduced coenzyme Q 10  represented by the following formula (1), and a composition for inhibiting arteriosclerosis containing both a cholesterol-lowering agent and said reduced coenzyme Q 10

[0001] This is a divisional of application Ser. No. 10/275,553 filedNov. 7, 2002; which is a §371 National Stage Application of PCTApplication No. PCT/JP01/03862 filed May 9, 2001, the disclosure ofwhich is incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to a method for achieving apotentiated effect of inhibiting arteriosclerosis expressed through theconcomitant use of a drub having cholesterol-lowering activity, forexample a hydroxymethylglutaryl-CoA reductase inhibitor (hereinafterreferred to as a statin), and a reduced coenzyme Q₁₀ occurring in plasmalow-density lipoprotein (hereinafter referred to a LDL) and having LDLantioxidant activity and to a composition therefor.

BACKGROUND ART

[0003] Coronary arterial disease is one of the diseases deserving theutmost attention today. It is suspected that a variety of factorscontribute in a complex way to arteriosclerosis which triggers coronaryarterial disease but an increased blood level of cholesterol is a majorfactor and the more direct factor is the increase and oxidativedegeneration of low-density lipoprotein (LDL) which comprisescholesterol particles.

[0004] For the prevention of arteriosclerosis, it is important to lowerthe blood cholesterol level. Statins reportedly produce markedcholesterol lowering effects in patients with hypercholesterolemia.Statins inhibit hydroxymethylglutaryl-CoA reductase, therate-determining enzyme in cholesterol biosynthesis, to therebyinterfere with cholesterol biosynthesis and through the consequentreduction in intrahepatic cholesterol content and ensuing increase inLDL receptors, lower the plasma cholesterol level. However,notwithstanding the fact that it is about a decade since this drub wasdeveloped, thee has been reportedly no change in the incidence ofconronary arterial disease and it is, thus, insufficient to merely lowercholesterol alone for the inhibition of arteriosclerosis.

[0005] Regarding the etiologic mechanism of arteriosclerosis, the LDLoxide hypothesis is known. Thus, it is postulated that LDL is oxidizedto ox-LDL, which is taken up in macrophages and the resulting foam cellsare deposited on the arterial wall to cause progression ofarteriosclerosis. Recent years have seen many research results endorsingthis hypothesis. The antiarteriosclerotic effect of theantihyperlipidemic drug probucol is considered to arise from itsantioxidant activity, not from its cholesterol-lowering activity. Thus,for the inhibition of arteriosclerosis, not only depression of thecholesterol level but also inhibition of the oxidation of LDL can be aneffective means.

[0006] As a natural antioxidant occurring in LDL, coenzyme Q₁₀ andvitamin E are known. Heretofore, in view of its abundant occurrence inLDL, vitamin E was once considered to be a central substance ofantioxidant activity but recent studies have revealed that coenzyme Q₁₀is a more important factor. While coenzyme Q₁₀ is a molecule synthesizedin vivo, it is well known that its biosynthetic pathway involveshydroxymethylglutaryl-CoA as does the biosynthetic pathway ofcholesterol and, therefore, the biosynthesis of coenzyme Q₁₀ is alsoinhibited by said statins. Actually many cases have been reported inwhich the administration of a statin caused depressions in the plasmacoenzyme Q₁₀ level. Moreover, the administration of a statin reportedlyrendered LDL more susceptible to oxidation and, therefore, the influenceof a statin on coenzyme Q₁₀ and, hence, on the oxidizability of LDL hasbeen suspected. To deal with the above decrease in coenzyme Q₁₀ causedby the administration of statin, attempts have been made to compensatefor the decrease by administering coenzyme Q₁₀(JP-A 02-233611, U.S. Pat.No. 5,082,650, U.S. Pat. No. 5,316,765).

[0007] Coenzyme Q₁₀ occurs in two forms, the oxidized form and thereduced form, and it is known that, in the living body, usually about 40to 90% of the coenzyme exists in the reduced form. It is the reducedcoenzyme Q₁₀ that exhibits antioxidant activity within LDL particles andregardless of how large its amount is, the oxidized coenzyme Q₁₀ doesnot expresses antioxidant activity at all. Therefore, for the inhibitionof arteriosclerosis, it is necessary to increase the reduced coenzymeQ₁₀ in the plasma. However, in the above-mentioned administration ofcoenzyme Q₁₀, the oxidized coenzyme Q₁₀ (ubiquinone) is invariably used.

SUMMARY OF THE INVENTION

[0008] The present invention has for its object to provide a method anda composition for achieving a more efficient recovery of the antioxidantactivity of plasma LDL, or even augmentation thereof, which has beendepressed by the use of an antihyperlipidemic or cholesterol-loweringdrug to thereby insure a potentiated antiarteriosclerotic efficacy.

[0009] The present inventors conducted intensive investigations toaccomplish the above object and found that compared with theadministration of oxidized coenzyme Q₁₀, the administration of thereduced coenzyme Q₁₀ causes a prominent increase in the plasmaconcentration of the reduced coenzyme Q₁₀. The present invention hasbeen accordingly accomplished.

[0010] The present invention, therefore, relates to a method forinhibiting arteriosclerosis, which comprises using a combination of acholesterol-lowering agent and the reduced coenzyme Q₁₀ represented bythe following formula (1).

[0011] The present invention further relates to an antiarterioscleroticcomposition comprising both of said cholesterol-lowering agent and saidreduced coenzyme Q₁₀.

[0012] It is known to employ the oxidized coenzyme Q₁₀ for the purposeof compensating for the decrease in plasma coenzyme Q₁₀ concentrationfollowing administration of a statin. However, the use of the reducedcoenzyme Q₁₀ according to the invention results in a marked elevation inthe plasma concentration of the reduced coenzyme Q₁₀ as compared withthe administration of the oxidized form and, therefore, is veryeffective in inhibiting oxidation of LDL and, hence, for the inhibitionof arteriosclerosis.

[0013] The expression “inhibition of arteriosclerosis” as used in thisspecification means both prophylaxis for preventing arteriosclerosis andtherapy for curing arteriosclerosis.

[0014] The present invention is now described in detail.

DISCLOSURE OF THE INVENTION

[0015] The method of inhibiting arteriosclerosis according to thepresent invention comprises using a cholesterol-lowering agent and thereduced coenzyme Q₁₀ in combination.

[0016] The cholesterol-lowering agent mentioned above is notparticularly restricted but includes a hydroxymethylglutaryl-CoAreductase inhibitor (statin), a clofibrate antihyperlipidemic drug, anicotinic acid derivative, a sterol derivative, elastase, apolyenephosphatidylcholine, melinamide, pantethine, icosapentanoicacid-EDTA, and so forth. However, among these, ahydroxymethylglutaryl-CoA reductase inhibitor is preferred.

[0017] The hydroxymethylglutaryl-CoA reductase inhibitor is notparticularly restricted but includes lovastatin, simvastatin,pravastatin, atorvastatin, rosuvastatin, fluvastatin, cerivastatin, andpitavastatin, among others.

[0018] The ratio of the cholesterol-lowering agent to the reducedcoenzyme Q₁₀ is not particularly restricted but is preferably 1:10 to10:1 by weight, more preferably 1:5 to 5:1 by weight.

[0019] In the method of the invention, the cholesterol-lowering agentand the reduced coenzyme Q₁₀ are preferably administered both orally,and the dosage based on the reduced coenzyme Q₁₀ is preferably 10 mg to1 g, more preferably 50 to 500 mg, in this oral regimen.

[0020] In the method of the invention, the cholesterol-lowering agentand the reduced coenzyme Q₁₀ may be administered at staggered times orconcurrently. In the concurrent administration, the cholesterol-loweringagent and the reduced coenzyme Q₁₀ may be administered as a singlepreparation comprising the two active agents or each as an independentpreparation. Furthermore, the start of administration of the reducedcoenzyme Q₁₀ may either precede or follow the start of administration ofthe cholesterol-lowering agent or may be simultaneous.

[0021] The technology of providing said reduced coenzyme Q₁₀ is notparticularly restricted but may for example comprise preparing coenzymeQ₁₀ by the known method, such as chemical synthesis, fermentation, orextraction from a naturally-occurring material, and concentrating theeluate containing the reduced coenzyme Q₁₀ by chromatography. Anotherversion of the technology comprises adding a conventional reducingagent, such a sodium borohydride, sodium dithionite (sodiumhydrosulfite) or the like, to above coenzyme Q₁₀ to reduce the oxidizedcoenzyme Q₁₀ to the reduced coenzyme Q₁₀ occurring in said coenzyme Q₁₀in the routine manner and concentrating the same by chromatography. Theobjective coenzyme Q₁₀ can also be obtained by permitting said reducingagent to act on the available high-purity coenzyme Q₁₀.

[0022] The antiarteriosclerotic composition of the present invention isprovided by formulating said reduced coenzyme Q₁₀ and acholesterol-lowering agent.

[0023] The cholesterol-lowering agent is not particularly restricted butis preferably a hydroxymethylglutaryl-CoA reductase inhibitor (a statin)

[0024] The formulating ratio of the cholesterol-lowering agent to thereduced coenzyme Q₁₀ is not particularly restricted but is preferably1:10 to 10:1, more preferably 1:5 to 5:1, by weight.

[0025] The composition of the invention may be supplemented with otherpharmaceutically acceptable formulating agents in suitable amounts inthe routine manner. Such pharmaceutical formulating agents are notparticularly restricted but include excipients, disintegrators,lubricants, binding agents, antioxidants, coloring agents,anticoagulants, absorption promoters, solubilizers for activeingredients, stabilizers, and so on.

[0026] The excipient mentioned above is not particularly restricted butincludes sucrose, lactose, glucose, cornstarch, mannitol, crystallinecellulose, calcium phosphate, and calcium sulfate, among others.

[0027] The disintegrator mentioned above is not particularly restrictedbut includes starch, agar, calcium citrate, calcium carbonate, calciumhydrogen carbonate, dextrin, crystalline cellulose,carboxymethylcellulose and tragacanth, among others.

[0028] The lubricant mentioned above is not particularly restricted butincludes, talc, magnesium stearate, polyethylene glycol, silica andhydrogenated vegetable oil, among others.

[0029] The binding agent mentioned above is not particularly restrictedbut includes ethylcellulose, methylcellulose,hydroxypropylmethylcellulose, tragacanth, shellac, gelatin, gum arabic,polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid,polymethacrylic acid and sorbitol, among others.

[0030] The antioxidant mentioned above is not particularly restrictedbut includes ascorbic acid, tocopherol, vitamin A, carotene, sodiumhydrogen sulfite, sodium thiosulfate, sodium pyrosulfite and citricacid, among others.

[0031] The coloring agent mentioned above is not particularly restrictedbut includes the authorized coloring agent for addition topharmaceutical products.

[0032] The anticoagulant mentioned above is not particularly restrictedbut includes stearic acid, talc, light silicic anhydride and silicicacid hydrate, among others.

[0033] The absorption promoter mentioned above is not particularlyrestricted but includes higher alcohols, higher fatty acids, andsurfactants such as glycerol fatty acid esters, among others.

[0034] The above-mentioned solubilizer for active ingredients is notparticularly restricted but includes benzoic acid, sodium benzoate,ethyl p-hydroxybenzoate, and so forth.

[0035] The usual dosage for an oral antiarteriosclerotic composition ofthe invention is preferably 10 mg to 1 g, more preferably 50 to 500 mg,based on coenzyme Q₁₀.

[0036] The composition of the invention can be provided in a variety ofdosage forms. For example, soft capsules can be produced by adding ordissolving the reduced coenzyme Q₁₀ and the cholesterol-lowering agentin natural oil, an oily higher fatty acid, a higher fatty acidmonoglyceride, or a mixture thereof and filling a soft capsule shellwith the resulting oily product. In this case, any of gelatin-basedcapsule materials and capsule materials based on other water-solublepolymers can be employed. The capsules include microcapsules as well.

BEST MODE FOR CARRYING OUT THE INVENTION

[0037] The following examples and pharmaceutical production examplesillustrate the present invention in further detail without defining thescope of the invention.

EXAMPLE 1 The Effect of the Reduced Coenzyme Q₁₀ and the OxidizedCoenzyme Q₁₀ on the Plasma Level of the Reduced Coenzyme Q₁₀ in Hamsterson High-Fat Diet

[0038] Hamsters were fed on a high-fat diet containing 1.5% ofcholesterol for one week and, then, a 1% (w/v) solution of either theoxidized or the reduced coenzyme Q₁₀ in olive oil was administeredorally for 4 consecutive days. After overnight fasting, the plasma wascollected and the reduced coenzyme Q₁₀ in the plasma was assayed byHPLC. TABLE 1 Plasma level of the reduced coenzyme Q₁₀ (n = 3) Gain inreduced coenzyme Q₁₀ Treatment in plasma (ng/ml) Administration ofoxidized 10.1 (100%) coenzyme Q₁₀ Administration of reduced 14.4 (143%)coenzyme Q₁₀

[0039] It can be seen from Table 1 that compared with the group giventhe oxidized coenzyme Q₁₀, the plasma level of reduced coenzyme Q₁₀ waselevated more prominently in the group given the reduced coenzyme Q₁₀.

EXAMPLE 2 Evaluation of the Antioxidant Activity of Plasma Low-DensityLipoprotein (LDL) in Hamsters on High-Fat Diet

[0040] Hamsters were fed on a high-fat diet containing 1.5% ofcholesterol for 1 week and, then, dosed orally with a 1% (w/v) solutionof either the oxidized or the reduced coenzyme Q₁₀ in olive oil and 10mg of pravastatin per kg body weight for 4 consecutive days. Afterovernight fasting, the plasma was collected, the plasma LDL wasseparated by density-gradient centrifugation method, and as a marker ofLDL antioxidant activity, the lag time was measured. The lag time isdefined herein as the value found by adding a copper ion (1 μM) to LDL(40 μg protein/mL) and monitoring at 234 nm the time course ofconjugated diene structure of oxidized fat/fatty acid resulting fromoxidation to determine the time interval until a sharp increase hadbegun in the conjugated diene concentration. The results are shown inTable 2. TABLE 2 LDL antioxidant activity (n = 3) Treatment Lag time(min) Solvent control 17 ± 4  Administration of oxidized 25 ± 8*coenzyme Q₁₀ Administration of reduced  44 ± 9** coenzyme Q₁₀

[0041] It can be seen from Table 2 that while administration of theoxidized coenzyme Q₁₀ enhanced the antioxidant activity of LDL,administration of the reduced coenzyme Q₁₀ led to a greater enhancementin the LDL antioxidant activity.

[0042] (Pharmaceutical Example 1)

[0043] An encapsulated pharmaceutical product containing ahydroxymethylglutaryl-CoA reductase inhibitor and the reduced coenzymeQ₁₀ was manufactured according to the following formula. Pravastatin 20mg weight parts Reduced coenzyme Q₁₀ 50 mg weight parts Lactose 200 mgweight parts  Magnesium stearate  5 mg weight parts

INDUSTRIAL APPLICABILITY

[0044] By using the method and composition of the invention, the plasmalevel of the reduced coenzyme Q₁₀ can be remarkably elevated topotentiate LDL antioxidant activity, leading to excellentantiarteriosclerotic efficacy.

1-4 (canceled).
 5. A method for enhancing LDL antioxidant activity which comprises administering to a subject a combination of a cholesterol-lowering agent and a reduced coenzyme Q₁₀ represented by the following formula (1).


6. The method according to claim 5, wherein the cholesterol-lowering agent is a hydroxymethylglutaryl-CoA reductase inhibitor. 